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. 2010 May 20;285(30):23105–23114. doi: 10.1074/jbc.M110.124990

FIGURE 2.

FIGURE 2.

Nutlin-arrested 2N and 4N cells enter S phase after Nutlin removal. A, HCT116, U2OS, A549, and MCF7 cells were either treated with Nutlin (Nut; 10 μmol/liter) for 24 h or treated with Nutlin for 24 h followed by Nutlin removal for an additional 16 h (+16h). BrdUrd incorporation by cells treated with Nutlin for 24 h was determined by adding BrdUrd (20 μm) to the culture medium 2 h before harvesting. BrdUrd incorporation after Nutlin removal was determined by culturing cells in medium containing BrdUrd (−Nutlin) for 16 h. Cells were harvested and stained with fluorescein isothiocyanate-conjugated anti-BrdUrd antibody and propidium iodide and analyzed by two-dimensional flow cytometry (BrdUrd versus propidium iodide/DNA content). Cells entering S phase from 2N state are labeled as S(2N); cells entering S phase from 4N state are labeled as S(4N). B, the percentage of 2N and 4N cells entering S phase after Nutlin removal was determined for all four cell lines. The percentage of 2N cells entering S phase was calculated by dividing the number of cells entering S phase S(2N) by the total number of 2N cells (S(2N)+2N). The percentage of 4N cells entering S phase was calculated by dividing the number of cells entering S phase S(4N) by the total number of 4N cells (S(4N)+4N). The mean of three independent experiments is shown ± S.E. (error bars).